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Complete integration of carbene-transfer chemistry into biosynthesis

Nature volume 617, pages 403–408 (2023)Cite this article

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Abstract

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule—styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

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Fig. 1: Carbene-transfer reactions for biosynthesis.
Fig. 2: Heterologous biosynthesis of the carbene precursor azaserine.
Fig. 3: Engineering cytochrome P450s to catalyse carbene-transfer reactions with azaserine as the carbene precursor.
Fig. 4: Biosynthesis of unnatural cyclopropanes by an abiological carbene-transfer reaction.

Data availability

The DNA sequences of plasmids used in this study have been deposited in the public version of the JBEI registry (http://public-registry.jbei.org). Accession codes are provided in Supplementary Table 2 (Part ID). The sequences and annotation of the aza BGC is available at GenBank (NZ_BEVZ01000003.1) spanning bases 82400–111549. The determined structure has been deposited in the PDB (8FBC). The MS proteomics data have been deposited at the ProteomeXchange Consortium through the PRIDE63 partner repository under the dataset identifier PXD037509. Source data are provided with this paper.

References

  1. Arnold, F. H. Directed evolution: bringing new chemistry to life. Angew. Chem. Int. Ed. Engl. 57, 4143–4148 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Wallace, S. & Balskus, E. P. Interfacing microbial styrene production with a biocompatible cyclopropanation reaction. Angew. Chem. Int. Ed. Engl. 54, 7106–7109 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Huang, J. et al. Unnatural biosynthesis by an engineered microorganism with heterologously expressed natural enzymes and an artificial metalloenzyme. Nat. Chem. 13, 1186–1191 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Davies, H. M. L. & Manning, J. R. Catalytic C-H functionalization by metal carbenoid and nitrenoid insertion. Nature 451, 417–424 (2008).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. Doyle, M. P., Duffy, R., Ratnikov, M. & Zhou, L. Catalytic carbene insertion into C-H bonds. Chem. Rev. 110, 704–724 (2010).

    Article  CAS  PubMed  Google Scholar 

  6. Ford, A. et al. Modern organic synthesis with α-diazocarbonyl compounds. Chem. Rev. 115, 9981–10080 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Zhu, D., Chen, L. F., Fan, H. L., Yao, Q. L. & Zhu, S. F. Recent progress on donor and donor-donor carbenes. Chem. Soc. Rev. 49, 908–950 (2020).

    Article  CAS  PubMed  Google Scholar 

  8. Kluger, R. Thiamin diphosphate: a mechanistic update on enzymic and nonenzymic catalysis of decarboxylation. Chem. Rev. 87, 863–876 (1987).

    Article  CAS  Google Scholar 

  9. Lee, J. K. & Houk, K. N. A proficient enzyme revisited: the predicted mechanism for orotidine monophosphate decarboxylase. Science 276, 942–945 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. Meyer, D., Neumann, P., Ficner, R. & Tittmann, K. Observation of a stable carbene at the active site of a thiamin enzyme. Nat. Chem. Biol. 9, 488–490 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Cochrane, A. R. et al. The natural product lepidiline A as an N-heterocyclic carbene ligand precursor in complexes of the type [Ir(cod)(NHC)PPh3)]X: synthesis, characterisation, and application in hydrogen isotope exchange catalysis. Catalysts 10, 161 (2020).

    Article  CAS  Google Scholar 

  12. Cheng, R. H. et al. Implications for an imidazole-2-yl carbene intermediate in the rhodanase-catalyzed C-S bond formation reaction of anaerobic ergothioneine biosynthesis. ACS Catal. 11, 3319–3334 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Coelho, P. S., Brustad, E. M., Kannan, A. & Arnold, F. H. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339, 307–310 (2013).

    Article  ADS  CAS  PubMed  Google Scholar 

  14. Brandenberg, O. F., Fasan, R. & Arnold, F. H. Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions. Curr. Opin. Biotechnol. 47, 102–111 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu, Z. & Arnold, F. H. New-to-nature chemistry from old protein machinery: carbene and nitrene transferases. Curr. Opin. Biotechnol. 69, 43–51 (2021).

    Article  CAS  PubMed  Google Scholar 

  16. Wallace, S. & Balskus, E. P. Designer micelles accelerate flux through engineered metabolism in E. coli and support biocompatible chemistry. Angew. Chem. Int. Ed. Engl. 55, 6023–6027 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wong, H. N. C. et al. Use of cyclopropanes and their derivatives in organic synthesis. Chem. Rev. 89, 165–198 (1989).

    Article  CAS  Google Scholar 

  18. Talele, T. T. The “cyclopropyl fragment” is a versatile player that frequently appears in preclinical/clinical drug molecules. J. Med. Chem. 59, 8712–8756 (2016).

    Article  CAS  PubMed  Google Scholar 

  19. Fulton, J. R., Aggarwal, V. K. & de Vicente, J. The use of tosylhydrazone salts as a safe alternative for handling diazo compounds and their applications in organic synthesis. Eur. J. Org. Chem. 2005, 1479–1492 (2005).

    Article  Google Scholar 

  20. Morandi, B. & Carreira, E. M. Iron-catalyzed cyclopropanation in 6 M KOH with in situ generation of diazomethane. Science 335, 1471–1474 (2012).

    Article  ADS  CAS  PubMed  Google Scholar 

  21. Zhang, L. M., DeMuynck, B. M., Paneque, A. N., Rutherford, J. E. & Nagib, D. A. Carbene reactivity from alkyl and aryl aldehydes. Science 377, 649–654 (2022).

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Xia, Y., Qiu, D. & Wang, J. B. Transition-metal-catalyzed cross-couplings through carbene migratory insertion. Chem. Rev. 117, 13810–13889 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Damiano, C., Sonzini, P. & Gallo, E. Iron catalysts with N-ligands for carbene transfer of diazo reagents. Chem. Soc. Rev. 49, 4867–4905 (2020).

    Article  CAS  PubMed  Google Scholar 

  24. Nawrat, C. C. & Moody, C. J. Natural products containing a diazo group. Nat. Prod. Rep. 28, 1426–1444 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Kawai, S. et al. Complete biosynthetic pathway of alazopeptin, a tripeptide consisting of two molecules of 6-diazo-5-oxo-l-norleucine and one molecule of alanine. Angew. Chem. Int. Ed. 60, 10319–10325 (2021).

    Article  CAS  Google Scholar 

  26. Le Maux, P., Nicolas, I., Chevance, S. & Simonneaux, G. Chemical reactivity of 6-diazo-5-oxo-l-norleucine (DON) catalyzed by metalloporphyrins (Fe,Ru). Tetrahedron 66, 4462–4468 (2010).

    Article  Google Scholar 

  27. Sugai, Y., Katsuyama, Y. & Ohnishi, Y. A nitrous acid biosynthetic pathway for diazo group formation in bacteria. Nat. Chem. Biol. 12, 73–75 (2016).

    Article  CAS  PubMed  Google Scholar 

  28. Ma, G. L. et al. Biosynthesis of tasikamides via pathway coupling and diazonium-mediated hydrazone formation. J. Am. Chem. Soc. 144, 1622–1633 (2022).

    Article  CAS  PubMed  Google Scholar 

  29. Bartz, Q. R. et al. Isolation and characterization of azaserine. Nature 173, 72–73 (1954).

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Stock, C. C., Clarke, D. A., Reilly, H. C., Rhoads, C. P. & Buckley, S. M. Azaserine, a new tumour-inhibitory substance: studies with crocker mouse sarcoma 180. Nature 173, 71–72 (1954).

    Article  ADS  CAS  PubMed  Google Scholar 

  31. Key, H. M. et al. Beyond iron: iridium-containing P450 enzymes for selective cyclopropanations of structurally diverse alkenes. ACS Cent. Sci. 3, 302–308 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lee, M. D. et al. New antitumor antibiotic, LL-D05139β. Fermentation, isolation, structure determination and biological activities. J. Antibiot. 40, 1657–1663 (1987).

    Article  CAS  Google Scholar 

  33. Matsuda, K. et al. Genome mining of amino group carrier protein-mediated machinery: discovery and biosynthetic characterization of a natural product with unique hydrazone unit. ACS Chem. Biol. 12, 124–131 (2017).

    Article  CAS  PubMed  Google Scholar 

  34. Twigg, F. F. et al. Identifying the biosynthetic gene cluster for triacsins with an N-hydroxytriazene moiety. ChemBioChem 20, 1145–1149 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Williams, M. V. & Tritz, G. J. Studies on the modes of action of azaserine inhibition of Escherichia coli. Potentiation of phenylalanine reversal. J. Antimicrob. Chemother. 3, 65–77 (1977).

    Article  CAS  PubMed  Google Scholar 

  36. Geisen, S. M. et al. Direct alkylation of deoxyguanosine by azaserine leads to O6-carboxymethyldeoxyguanosine. Chem. Res. Toxicol. 34, 1518–1529 (2021).

    Article  CAS  PubMed  Google Scholar 

  37. Gober, J. G. et al. P450-mediated non-natural cyclopropanation of dehydroalanine-containing thiopeptides. ACS Chem. Biol. 12, 1726–1731 (2017).

    Article  CAS  PubMed  Google Scholar 

  38. Zhang, R. J. K. et al. Enzymatic assembly of carbon-carbon bonds via iron-catalysed sp3 C-H functionalization. Nature 565, 67–72 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  39. Nguyen, K. T. et al. Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam. FEBS Open Bio 11, 124–132 (2021).

    Article  CAS  PubMed  Google Scholar 

  40. Ost, T. W. B. et al. Oxygen activation and electron transfer in flavocytochrome P450BM3. J. Am. Chem. Soc. 125, 15010–15020 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. McKenna, R. & Nielsen, D. R. Styrene biosynthesis from glucose by engineered E. coli. Metab. Eng. 13, 544–554 (2011).

    Article  CAS  PubMed  Google Scholar 

  42. McKenna, R., Thompson, B., Pugh, S. & Nielsen, D. R. Rational and combinatorial approaches to engineering styrene production by Saccharomyces cerevisiae. Microb. Cell Fact. 13, 123 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Lee, K., Bang, H. B., Lee, Y. H. & Jeong, K. J. Enhanced production of styrene by engineered Escherichia coli and in situ product recovery (ISPR) with an organic solvent. Microb. Cell Fact. 18, 79 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Lin, F. M., Ferguson, K. L., Boyer, D. R., Lin, X. X. N. N. & Marsh, E. N. G. Isofunctional enzymes PAD1 and UbiX catalyze formation of a novel cofactor required by ferulic acid decarboxylase and 4-hydroxy-3-polyprenylbenzoic acid decarboxylase. ACS Chem. Biol. 10, 1137–1144 (2015).

    Article  CAS  PubMed  Google Scholar 

  45. Brandenberg, O. F., Chen, K. & Arnold, F. H. Directed evolution of a cytochrome P450 carbene transferase for selective functionalization of cyclic compounds. J. Am. Chem. Soc. 141, 8989–8995 (2019).

    Article  CAS  PubMed  Google Scholar 

  46. Fischer, M. & Sawers, R. G. A universally applicable and rapid method for measuring the growth of streptomyces and other filamentous microorganisms by methylene blue adsorption-desorption. Appl. Environ. Microbiol. 79, 4499–4502 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  47. Aziz, R. K. et al. The RAST server: rapid annotations using subsystems technology. BMC Genom. 9, 75 (2008).

    Article  Google Scholar 

  48. Navarro-Munoz, J. C. et al. A computational framework to explore large-scale biosynthetic diversity. Nat. Chem. Biol. 16, 60–68 (2020).

    Article  CAS  PubMed  Google Scholar 

  49. Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

    Article  CAS  PubMed  Google Scholar 

  50. Winter, G., Lobley, C. M. C. & Prince, S. M. Decision making in xia2. Acta Crystallogr. D 69, 1260–1273 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Mccoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  53. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).

    Article  CAS  Google Scholar 

  54. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  56. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  PubMed  Google Scholar 

  57. Lee, T. S. et al. BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J. Biol. Eng. 5, 12 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Yuzawa, S. et al. Short-chain ketone production by engineered polyketide synthases in Streptomyces albus. Nat. Commun. 9, 4569 (2018).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  59. Ehrlich, J. et al. Process for producing azaserine. US patent 2996435A (1961).

  60. Chen, Y., Gin, J. & Petzold, C. Discovery proteomic (DIA) LC-MS/MS data acquisition and analysis V.2. Protocols.io https://doi.org/10.17504/protocols.io.e6nvwk1z7vmk/v2 (2022).

  61. Demichev, V., Messner, C. B., Vernardis, S. I., Lilley, K. S. & Ralser, M. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat. Methods 17, 41–44 (2020).

    Article  CAS  PubMed  Google Scholar 

  62. Ahrne, E., Molzahn, L., Glatter, T. & Schmidt, A. Critical assessment of proteome-wide label-free absolute abundance estimation strategies. Proteomics 13, 2567–2578 (2013).

    Article  CAS  PubMed  Google Scholar 

  63. Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50, D543–D552 (2022).

    Article  CAS  PubMed  Google Scholar 

  64. Madeira, F. et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res. 50, W276–W279 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by Joint BioEnergy Institute (https://www.jbei.org), which is supported by the DOE, Office of Science, Office of Biological and Environmental Research under contract DE-AC02-05CH11231 and National Science Foundation grant 2027943. We thank H. Celik and the staff at UC Berkeley’s NMR facility in the College of Chemistry (CoC-NMR) for spectroscopy assistance. Instruments in the CoC-NMR are supported in part by NIH S10OD024998.

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Authors and Affiliations

  1. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jing Huang, Pablo Cruz-Morales, Ramu Kakumanu, Edward E. K. Baidoo, Qingyun Dan, Yan Chen, Christopher J. Petzold, Aindrila Mukhopadhyay & Jay D. Keasling

  2. Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA

    Jing Huang, Pablo Cruz-Morales, Kai Deng, Jose Henrique Pereira, Ramu Kakumanu, Edward E. K. Baidoo, Qingyun Dan, Yan Chen, Christopher J. Petzold, Trent R. Northen, Paul D. Adams, Aindrila Mukhopadhyay & Jay D. Keasling

  3. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA

    Jing Huang, Douglas S. Clark & Jay D. Keasling

  4. Department of Chemistry, University of California, Berkeley, CA, USA

    Andrew Quest & John F. Hartwig

  5. Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Andrew Quest & John F. Hartwig

  6. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark

    Pablo Cruz-Morales & Jay D. Keasling

  7. Department of Biomaterials and Biomanufacturing, Sandia National Laboratories, Livermore, CA, USA

    Kai Deng

  8. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jose Henrique Pereira, Paul D. Adams & Douglas S. Clark

  9. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Devon Van Cura & Emily P. Balskus

  10. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Trent R. Northen & Aindrila Mukhopadhyay

  11. Department of Bioengineering, University of California, Berkeley, CA, USA

    Paul D. Adams & Jay D. Keasling

  12. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Emily P. Balskus

  13. Synthetic Biochemistry Center, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China

    Jay D. Keasling

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  1. Jing Huang
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Contributions

J.H. and J.D.K. conceived the project. J.H. and J.D.K. wrote the manuscript with input from all of the other authors. J.D.K., A.M., J.F.H. and D.S.C. provided project guidance. A.Q. and K.D. synthesized the chemical standards. J.H., P.C.-M. and D.V.C. identified the gene cluster. J.H.P. determined the crystal structure. Q.D. purified the proteins. J.H., R.K. and E.E.K.B. performed MS. Y.C. performed proteomics analysis. J.H. conducted the other experiments. E.P.B., P.D.A., T.R.N. and C.J.P. provided resources. All of the authors contributed to the manuscript.

Corresponding authors

Correspondence to Douglas S. Clark, John F. Hartwig, Aindrila Mukhopadhyay or Jay D. Keasling.

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Competing interests

J.D.K. has a financial interest in Amyris, Demetrix, Maple Bio, Lygos, Napigen, Berkeley Yeast, Zero Acre Farms, Ansa Biotechnologies, Apertor Pharmaceuticals, ResVit Bio and Cyklos Materials. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Effect of Na2S2O4 on the activity of haemin and Ir(Me)MPIX for the reaction of styrene with azaserine.

a, Addition of Na2S2O4 decreased the reaction yield when using Ir(Me)MPIX as catalyst. TON, turn-over number. Reaction conditions are described in Fig. 2 legend. Data are mean value for 2 reaction replicates. b, Na2S2O4 is necessary for the activity of haemin toward the reaction. EIC ([M+H]+, m/z 250.1074) for target products. The traces are representative of two reaction replicates. The reaction contained 5 mM styrene, 5 mM azaserine, 10 µM haemin or no catalyst, 0 or 10 mM Na2S2O4, 5 vol% ethanol, and M9-N buffer and was conducted at 22 °C under aerobic conditions for 18 h. Standard, chemically synthesized authentic standard mixture of the four diastereomers.

Source data

Extended Data Fig. 2 Azaserine toxicity on E. coli and S. albus.

a, Azaserine is toxic to E. coli BL21(DE3). b, Azaserine does not affect the growth of S. albus under tested concentrations. Biomass was normalized to that of culture without addition of azaserine. Data are mean ± s.d.; n = 3 biological replicates.

Source data

Extended Data Fig. 3 Expression of azaserine gene cluster from S. fragilis in S. albus.

a, Bioinformatic annotation of the azaserine gene cluster and comparison with the biosynthetic gene clusters for some natural N–N bond-containing compounds. b, Proteomic analysis of the azaserine gene cluster when expressed in S. albus. Data are mean ± s.d.; n = 3 biological replicates.

Source data

Extended Data Fig. 4 Differences in azaserine degradation in various media.

a, Azaserine titre continues to decrease after removal of S. albus cells. The azaserine-producing S. albus was grown in TSB medium. After 24 h, the cells were removed from culture broth using a 0.22-µm sterile filter, the filtrate was incubated at 30 °C (labelled as 0 h), and the azaserine concentration was monitored at different time points. b, Azaserine is stable in fresh TSB medium of normal pH 7.3 or adjusted pH 8.4. Equal volume of azaserine stock was added to a final concentration of about 35 mg/L at 0 h. Data are mean ± s.d.; n = 3 biological (a) or technical (b) replicates.

Source data

Extended Data Fig. 5 Purified P450-T2 WT and mutant catalysing the reaction of styrene with azaserine in vitro.

a, Coomassie Blue stained SDS-PAGE gel of purified protein P450-T2 WT (left) and P450-T2-5 mutant (right). Each lane is a sample from fractions collected during ion exchange purification. b, Purified P450-T2 WT and P450-T2-5 mutant proteins catalyse the reaction in vitro with high diastereoselectivity. 5th, P450-T2-5 mutant. Reaction conditions: 5 mM styrene, 5 mM azaserine, 10 µM enzyme, 10 mM Na2S2O4, 5 vol% ethanol, M9-N buffer, conducted at 22 °C under aerobic condition for 18 h. Ptotal, sum area for all diastereomers. Grey bars indicate the dr. Data are mean ± s.d.; n = 3 reaction replicates.

Source data

Extended Data Fig. 6 CYP203A1 WT and axial ligand mutants for the reaction of styrene with azaserine.

EIC ([M+H]+, m/z 250.1074) for target products. Representative traces for two repeated experiments. The reactions contained 5 mM styrene, 5 mM azaserine, E. coli cells with concentration of 30 OD600 as catalyst, 5 vol% ethanol, and M9-N buffer and were conducted at 22 °C under aerobic conditions for 18 h.

Extended Data Fig. 7 Conserved amino acids selected for saturation mutagenesis.

The residue labelled in blue is the haem ligand cysteine in those P450s. Residues labelled in red are two conserved residues previously reported to affect the catalytic behaviour in P450 BM3. Sequences were aligned with Clustal Omega64.

Extended Data Fig. 8 Exploration of the conditions for styrene biosynthesis in S. albus.

a, Heterologously expressing PAL2 and FDC1 are sufficient to generate styrene. pAZA121 and pAZA138 are two integration plasmids used to introduce the styrene pathway into S. albus. b, Production of styrene by engineered S. albus grown in TSB medium supplemented with additional 0 mM, 2 mM or 4 mM Phe. Data are mean ± s.d.; n = 3 biological samples.

Source data

Extended Data Fig. 9 Characterization and time course of accumulation of the final products.

a, MS/MS (20 eV) spectra of the P1 standard (red) and the biosynthesized major product (black). b, Biosynthesis of final products during the 96-h fermentation process. The 24-h data are not presented because small quantities of product were observed (<10 µg/L), and thus the titre could not be accurately calculated. 1B medium with 4 mM Phe was used to generate the final products. Data are mean ± s.d.; n = 3 biological samples.

Source data

Extended Data Table 1 Screened sites for directed evolution of P450-T2
Full size table

Supplementary information

Supplementary Information

Supplementary Tables 1–3 (DNA and proteins sequences, synthesis of compounds), Supplementary Figs. 1–38 and Supplementary References.

Reporting Summary

Source data

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Huang, J., Quest, A., Cruz-Morales, P. et al. Complete integration of carbene-transfer chemistry into biosynthesis. Nature 617, 403–408 (2023). https://doi.org/10.1038/s41586-023-06027-2

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  • DOI: https://doi.org/10.1038/s41586-023-06027-2

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